Characterization of a natural heterodimer between MLV genomic RNA and the SD′ retroelement generated by alternative splicing

Abstract

Murine leukemia virus (MLV) specifically packages both genomic RNA (FL RNA) and a subgenomic RNA, which we call SD9. SD9 RNA results from alternative splicing of FL RNA. It is reverse-transcribed, and its DNA copy, integrated into the host genome, constitutes a splice donor-associated retroelement. FL and SD9 RNAs share a common 59-UTR that includes the packaging/dimerization signal (Psi). To investigate whether the mechanism of copackaging of these two RNAs involves RNA heterodimerization, we examined the spontaneous dimerization capacity of the two RNAs as large synthetic RNAs transcribed in vitro. We showed that SD9 RNA not only formed homodimers with similar efficiency as the FL RNA, but that FL and SD9 RNAs also formed FL/SD9 heterodimers via Psi sequences. Comparison of the thermostabilities determined for these different dimeric species and competition experiments with Psi RNA fragments indicate the recruitment of similar dimer-linkage interactions within the Psi region. To validate these results, the dimeric state of the SD9 RNA was analyzed in MLV particles. RNA capture assays performed with the FL RNA as bait revealed that SD9, and not the host packageable U6 or 7SL RNAs, was associated with the FL RNA in virions. Heterodimerization of SD9 RNA with FL RNA may argue for the recent concept of a nuclear dimerization at or near the site of transcription and raises the new hypothesis of RNA dimerization during splicing. Furthermore, FL/SD9 heterodimerization may have leukemogenic consequences by influencing the pool of genomic dimers that will undergo recombinogenic template switching by reverse transcriptase.

Retroviral RNA Dimerization: From Structure to Functions

“…Indeed, heterodimerization is enhanced when two proviruses are spatially close, suggesting that MuLV gRNA dimerization is coupled to transcription and splicing processes ( Maurel and Mougel, 2010 ). Consistent with these observations, MuLV RNAs transcribed from the same locus form dimers at high frequencies ( Flynn et al, 2004 ; Kharytonchyk et al, 2005 ; Rasmussen and Pedersen, 2006 ; Maurel et al, 2007 ). Interestingly, mutations of RNA elements involved in dimerization or packaging processes impact the intracellular transport of viral genome and result in aberrant accumulation in the nucleus or in the cytoplasm ( Basyuk et al, 2005 ; Smagulova et al, 2005 ).…”

Murine leukemia virus RNA dimerization is coupled to transcription and splicing processes

“…Similarly, nascent RNAs that are undergoing synthesis might adopt a more favorable conformation for dimerization compared to complete transcripts. In support of this model, dimerization occurred more efficiently for large synthetic MLV or HIV RNAs during in vitro transcription than post-synthesis [30,36,49]. Alternatively, co-transcription and splicing could enhance dimerization by providing high local RNA concentration in a subnuclear domain that facilitates RNA-RNA interactions.…”

Cell biology of retroviral RNA packaging

“…81,82 Later studies performed on MLV and HIV-1 particles generated in mammalian cells have demonstrated that, in the absence of viral gRNA, VLPs specifically package cellular mRNAs randomly, as mRNA species detected in VLPs are in proportion to their cellular levels, 24,83,84 at the exception of some RNA species such as 7SL, and U6 RNAs, that are specifically enriched in wild-type HIV-1 and MLV particles and whose function remains unclear. 24,25,83,84 Interestingly, retroviral cores containing either viral or cellular RNAs were disassembled by RNase treatment, 83 confirming the importance of the RNA-Gag interaction in the formation and structure of retroviral particles.…”

“…Nevertheless, viral spliced RNAs are also selectively packaged in infectious particles and subsequently reverse-transcribed in infected cells. [22][23][24][25][26][27] Although the molecular mechanisms that govern RNA packaging selectivity remain unclear, initial evidence has shown that HIV-1 incorporates the gRNA and the spliced mRNA species in a competitive manner. 24 The selective recruitment of the gRNA is at least in part the result of the high affinity binding of Psi sequences for GagNC protein.…”